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J. Biol. Chem., Vol. 283, Issue 26, 18076-18085, June 27, 2008

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N-type Inactivation of the Potassium Channel KcsA by the Shaker B "Ball" Peptide

MAPPING THE INACTIVATING PEPTIDE-BINDING EPITOPE^*

M. Luisa Molina¹², Francisco N. Barrera¹³, José A. Encinar, M. Lourdes Renart², Asia M. Fernández, José A. Poveda, Jorge Santoro, Marta Bruix, Francisco Gavilanes^¶, Gregorio Fernández-Ballester, José L. Neira^||, and José M. González-Ros⁴

From the Instituto de Biología Molecular y Celular, Universidad Miguel Hernández, 03202 Elche (Alicante), the Instituto de Química-Física Rocasolano, CSIC, 28006 Madrid, the ^¶Departamento de Bioquímica, Facultad de Ciencias Químicas, Universidad Complutense, 28040 Madrid, and the ^||Biocomputation and Complex Systems Physics Institute, 50009 Zaragoza, Spain

The effects of the inactivating peptide from the eukaryotic Shaker BK⁺ channel (the ShB peptide) on the prokaryotic KcsA channel have been studied using patch clamp methods. The data show that the peptide induces rapid, N-type inactivation in KcsA through a process that includes functional uncoupling of channel gating. We have also employed saturation transfer difference (STD) NMR methods to map the molecular interactions between the inactivating peptide and its channel target. The results indicate that binding of the ShB peptide to KcsA involves the ortho and meta protons of Tyr⁸, which exhibit the strongest STD effects; the C4H in the imidazole ring of His¹⁶; the methyl protons of Val⁴, Leu⁷, and Leu¹⁰ and the side chain amine protons of one, if not both, the Lys¹⁸ and Lys¹⁹ residues. When a noninactivating ShB-L7E mutant is used in the studies, binding to KcsA is still observed but involves different amino acids. Thus, the strongest STD effects are now seen on the methyl protons of Val⁴ and Leu¹⁰, whereas His¹⁶ seems similarly affected as before. Conversely, STD effects on Tyr⁸ are strongly diminished, and those on Lys¹⁸ and/or Lys¹⁹ are abolished. Additionally, Fourier transform infrared spectroscopy of KcsA in presence of ¹³C-labeled peptide derivatives suggests that the ShB peptide, but not the ShB-L7E mutant, adopts a β-hairpin structure when bound to the KcsA channel. Indeed, docking such a β-hairpin structure into an open pore model for K⁺ channels to simulate the inactivating peptide/channel complex predicts interactions well in agreement with the experimental observations.

Received for publication, December 12, 2007 , and in revised form, April 7, 2008.

^* This work was supported by Spanish Ministerio de Educación y Ciencia Grants CTQ2005-00360/BQU (to J. L. N.) and BFU2005-00749 (to J. M. G.-R.); FIPSE Experiment 36557/06 (to J. L. N.) and Grant BANCAJA-UMH IP/UR/01; and Consellería de Empresa, Universidad y Ciencia de la Generalitat Valenciana Grant GV07/017 (to J. A. E.). The costs of publication of this article were defrayed in part by the payment of page charges. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. Section 1734 solely to indicate this fact.

¹ These authors contributed equally to this work.

² Recipient of a predoctoral fellowship from the Generalitat Valenciana.

³ Supported in part by predoctoral fellowships from the Ministerio de Educación y Ciencia of Spain.

⁴ To whom correspondence should be addressed: Instituto de Biología Molecular y Celular, Edificio Torregaitán, Universidad Miguel Hernández, 03202 Elche (Alicante), Spain. Tel.: 34-966658757; Fax: 34-966658758; E-mail: gonzalez.ros{at}umh.es.

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